Thin-layer identification method of semen aesculi formula granules

Abstract

The invention discloses a thin-layer identification method of semen aesculi formula granules, which comprises the following steps: firstly extracting semen aesculi medicinal materials or formula granules thereof and semen aesculi reference medicinal materials by using petroleum ether, discarding the petroleum ether, adding water for dissolving or decocting, and then extracting by using ethyl acetate to obtain a test solution and a reference medicinal material solution; and respectively dispensing the reference medicinal material solution and the test solution on a thin-layer plate, developing by using a developing solvent, spraying a color developing agent, heating until the color of spots is clear, and carrying out comparison and identification under sunlight. According to the method, the sample is treated by adopting a simple treatment method and is inspected under the sunlight, the number of spots is large, the separation degree is relatively good, various components in the sample can be detected, and the specificity of the method is greatly improved. The method can be used for easily distinguishing the semen aesculi and counterfeit products thereof, can also be used for distinguishing different base sources of the semen aesculi, and has a relatively high practical value.

Description

Technical Field

[0001] The present invention relates to the quality identification of traditional Chinese medicines, and specifically relates to a thin-layer identification method for semen aesculi formula granules. Background Art

[0002] Semen aesculi is the dried and mature seed of the plants Aesculus chinensis Bge., Aesculus chinensis Bge. var. chekiangensis (Hu et Fang) Fang or Aesculus wilsonii Rehd. of the family Hippocastanaceae. It is harvested in autumn when the fruits are mature, the pericarp is removed, and then dried in the sun or by low-temperature drying. Semen aesculi is mainly produced in places such as Zhejiang, Jiangsu, and Henan. According to traditional Chinese medicine theory, it has the effects of soothing the liver and regulating qi, and harmonizing the stomach and relieving pain. The chemical components in semen aesculi mainly include triterpenoid saponins, flavonoids, coumarins, indole alkaloids, and fatty oils, etc.

[0003] Traditional Chinese medicine formula granules are a breakthrough in the application form of traditional Chinese medicine decoction pieces. Usually, they are made from decoction pieces as raw materials through standardized extraction (generally mainly water extraction), concentration, drying, and granulation. Their nature, flavor, meridian tropism, and efficacy are basically the same as those of the original decoction pieces, and they also have the advantages of not requiring decoction, being easy to dispense, store, and convenient to take, and are gradually widely used. In the first part of the Chinese Pharmacopoeia 2020 edition under semen aesculi, there is no thin-layer identification item for semen aesculi medicinal materials and semen aesculi formula granules. Moreover, since traditional Chinese medicine formula granules are extracted with water, their fat-soluble components are less, and the thin-layer identification method for medicinal materials is not applicable to formula granules. Therefore, establishing a rapid, simple, and low-cost thin-layer identification method for semen aesculi formula granules is an important topic. Summary of the Invention

[0004] Object of the Invention: Aiming at the deficiencies and defects of the prior art, the present invention provides a thin-layer identification method for semen aesculi medicinal materials or their formula granules. This method is simple, rapid in operation, has good chromatographic spot resolution, and low detection cost.

[0005] Technical Solution: The thin-layer identification method for semen aesculi medicinal materials or their formula granules described in the present invention includes the following steps:

[0006] The semen aesculi medicinal materials or their formula granules and the semen aesculi control medicinal materials are first extracted with petroleum ether, the petroleum ether is discarded, and after dissolving or decocting with water, they are then extracted with ethyl acetate to obtain the test solution and the control medicinal material solution;

[0007] The control medicinal material solution and the test solution are respectively spotted on a thin-layer plate, developed with a developing agent, sprayed with a color-developing agent, heated until the spots are clearly developed, and compared and identified under daylight.

[0008] The thin-layer identification method for the Chinese medicine material of Aesculus chinensis Bunge or its formula granules specifically includes the following steps:

[0009] (1) Take the control Chinese medicine material of Aesculus chinensis Bunge, add petroleum ether, shake or ultrasonicate, let stand, discard the supernatant, dry the residue by volatilizing the solvent, add water, heat and decoct, cool, centrifuge, take the supernatant and concentrate it, extract with ethyl acetate by shaking or ultrasonication, evaporate the ethyl acetate extract to dryness, dissolve the residue in an organic solvent to obtain the control Chinese medicine material solution;

[0010] (2) Take the Chinese medicine material of Aesculus chinensis Bunge, add petroleum ether, shake or ultrasonicate, let stand, discard the supernatant, dry the residue by volatilizing the solvent, add water, heat and decoct, cool, centrifuge, take the supernatant and concentrate it, extract with ethyl acetate by shaking or ultrasonication, evaporate the ethyl acetate extract to dryness, dissolve the residue in an organic solvent to obtain the test sample solution of the Chinese medicine material;

[0011] Or, take the formula granules of Aesculus chinensis Bunge, add petroleum ether, shake or ultrasonicate, let stand, discard the supernatant, dry the residue by volatilizing the solvent, dissolve in water, extract with ethyl acetate by shaking or ultrasonication, evaporate the ethyl acetate extract to dryness, dissolve the residue in an organic solvent to obtain the test sample solution of the formula granules; preferably, take the formula granules of Aesculus chinensis Bunge, grind them finely, and then add petroleum ether.

[0012] (3) Take the control Chinese medicine material solution and the test sample solution and spot them on a thin-layer plate respectively. After developing with the developing agent, spray with the color-developing agent, heat until the spots are clearly developed, and conduct comparative identification under daylight.

[0013] For the thin-layer identification method for the Chinese medicine material of Aesculus chinensis Bunge or its formula granules, the order of steps (1) and (2) can be interchanged.

[0014] For the thin-layer identification method for the Chinese medicine material of Aesculus chinensis Bunge or its formula granules, the organic solvent includes alcohol solvents. Preferably, the organic solvent includes methanol.

[0015] For the thin-layer identification method for the Chinese medicine material of Aesculus chinensis Bunge or its formula granules, the mass-volume ratio of the Chinese medicine material of Aesculus chinensis Bunge, the control Chinese medicine material of Aesculus chinensis Bunge or the formula granules of Aesculus chinensis Bunge to petroleum ether is 1:10 g / mL - 100 g / mL.

[0016] For the thin-layer identification method for the Chinese medicine material of Aesculus chinensis Bunge or its formula granules, the developing agent is a mixed solution of toluene, ethyl acetate, methanol, and formic acid.

[0017] For the thin-layer identification method for the Chinese medicine material of Aesculus chinensis Bunge or its formula granules, the volume ratio of toluene - ethyl acetate - methanol - formic acid in the developing agent is 8 - 12:1 - 3:1 - 3:0.2 - 0.8. Preferably, the developing agent is a mixed solution of toluene, ethyl acetate, methanol, and formic acid with a volume ratio of 10:2:2:0.5.

[0018] In the thin layer identification method of the sauerkraut medicinal material or its formula particles, the color developer is a 5-15% phosphomolybdic acid ethanol solution.

[0019] The thin layer identification method of the Sauerkraut medicinal material or its formula granules, the thin layer plate is a silica gel G thin layer plate.

[0020] In the thin layer identification method of the sauerkraut medicinal material or its formula granules, the heating until the spots are clearly colored is heating at 80-150° C. until the spots are clearly colored.

[0021] Technical points of the present invention:

[0022] 1. Remove the grease with petroleum ether first, dissolve it with water, and then shake it with ethyl acetate to extract the effective ingredients while removing the impurities. At the same time, it is easy to operate, has low inspection cost, and has high application value in actual production.

[0023] 2. Use reference medicinal materials as reference substances, use toluene-ethyl acetate-methanol-formic acid (10:2:2:0.5) as developing agent, spray with color developer and inspect under sunlight. The number of spots is large and the separation is good. It can detect multiple components in the sample, which greatly improves the specificity of the method.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. It solves the problem that there is no thin-layer identification item for Sago asiatica in the pharmacopoeia. 2. It solves the problem that the thin-layer identification method for Sago asiatica medicinal materials in the literature is not suitable for formula granules. 3. Using control medicinal materials as control substances, the samples are treated with simple processing methods and inspected under sunlight. The number of spots is large and the separation is good. Multiple components in the sample can be detected, which greatly improves the specificity of the method. 4. This method can easily distinguish Sago asiatica and its counterfeits (European horse chestnut, Yunnan horse chestnut), and at the same time, it can also distinguish different sources of Sago asiatica, which has great practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The chromatograms of the formula granules (23040049) of Salacia chinensis (Tianshi Li) obtained by different extraction methods and the reference medicinal materials of Salacia chinensis (Tianshi Li); 1: Test method 1; 2: Test method 2; 3: Test method 3; 4: Test method 4; 5: Test method 5; 6: Test method 6; 7: Test method 7; 8: Test method 8; S1: Salacia chinensis (Tianshi Li) reference medicinal materials (method 1); S2: Salacia chinensis (Tianshi Li) reference medicinal materials (method 2);

[0026] Figure 2It is the TLC chromatogram of different sample amounts of semen aesculi formula granules and semen aesculi reference medicinal materials; 1: 1 μl of test sample; 2: 2 μl of test sample; 3: 5 μl of test sample; 4: 10 μl of test sample; S1: 1 μl of reference medicinal material; S2: 2 μl of reference medicinal material; S3: 5 μl of reference medicinal material; S4: 10 μl of reference medicinal material;

[0027] Figure 3 It is the TLC chromatogram of semen aesculi formula granules and their negative control; 1-3: semen aesculi formula granules (23040049); 4: negative control; S: semen aesculi reference medicinal material;

[0028] Figure 4 It is the TLC chromatogram of semen aesculi formula granules under different temperature conditions; 1-3: semen aesculi formula granules (23040049); S: semen aesculi reference medicinal material;

[0029] Figure 5 It is the TLC chromatogram of semen aesculi formula granules under different humidity conditions; 1-3: semen aesculi formula granules (23040049); S: semen aesculi reference medicinal material;

[0030] Figure 6 It is the TLC chromatogram of semen aesculi formula granules on thin layer plates from different manufacturers; 1-3: semen aesculi formula granules (23040049); S: semen aesculi reference medicinal material;

[0031] Figure 7 It is the thin layer identification chromatogram of semen aesculi formula granules from different batches; 1: semen aesculi formula granules (23040049); 2: semen aesculi formula granules (23040059); 3: semen aesculi formula granules (23040069); S: semen aesculi reference medicinal material;

[0032] Figure 8 It is the thin layer identification chromatogram of semen aesculi formula granules, semen aesculi chinensis formula granules and semen aesculi wilsonii formula granules; 1-3: semen aesculi chinensis formula granules (2308038, 2308061, 2308062); 4-6: semen aesculi wilsonii formula granules (2407050, 2407051, 2407052); 7-9: semen aesculi formula granules (23040049, 23040059, 23040069); S: semen aesculi reference medicinal material;

[0033] Figure 9It is the thin-layer identification chromatogram of semen aesculi formula granules, semen hippocastani formula granules, and semen aesculi yunnanensis formula granules; 1-3: semen aesculi formula granules (23040049, 23040059, 23040069); 4-6: semen hippocastani formula granules (2307022, 2307023, 2307024); 7-9: semen aesculi yunnanensis formula granules (2308040, 2308063, 2308064); S: semen aesculi reference medicinal material;

[0034] Figure 10 It is the literature method 1 of semen aesculi granules; among which 1: semen aesculi formula granules (23040049);

[0035] Figure 11 It is the literature method 2 of semen aesculi granules; among which 1: semen aesculi formula granules (23040049);

[0036] Figure 12 It is the identification of esculetin in the literature method of semen aesculi granules; 1: semen aesculi formula granules (23040049); T: esculetin reference substance;

[0037] Figure 13 It is the test result with the developing solvent ethyl acetate - methanol - formic acid (10:1:0.5); among which, 1: semen aesculi formula granules (23040049); 2: semen aesculi formula granules (23040059); 3: semen aesculi formula granules (23040069); S: semen aesculi reference medicinal material. Specific implementation method

[0038] 1. Instruments and reagents

[0039] Instruments: Thin-layer automatic imager (CAMAG TLC VISUALIZER), ten-thousandth balance (METTLER TOLEDO), KQ-250E type ultrasonic cleaner (Kunshan Ultrasonic Electronics Co., Ltd.), silica gel G thin-layer plate (Qingdao Ocean Chemical Co., Ltd., Qingdao Kangyexin Medicinal Silica Gel Desiccant Co., Ltd., Yantai Xincheng Silica Gel Materials Co., Ltd.).

[0040] Test drugs: Methanol (Sinopharm Chemical Reagent Co., Ltd.), Ethanol (Sinopharm Chemical Reagent Co., Ltd.), Petroleum ether (30 - 60 °C) (Sinopharm Chemical Reagent Co., Ltd.), Ethyl acetate (Sinopharm Chemical Reagent Co., Ltd.), Formic acid (Sinopharm Chemical Reagent Co., Ltd.), Toluene (Shanghai Lingfeng Chemical Reagent Co., Ltd.) are all of analytical purity, and water; Semen Aesculi (Tian Shili) reference crude drug (Batch number: YC1012303042), Semen Aesculi (Tian Shili) formula granules (Batch numbers: 23040049, 23040059, 23040069) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0041] 2. Experimental methods

[0042] 2.1 Thin-layer identification conditions

[0043] Use a silica gel G thin-layer plate, with toluene - ethyl acetate - methanol - formic acid (10:2:2:0.5) as the developing agent, develop, take out, air dry, spray with 10% ethanolic solution of phosphomolybdic acid, heat at 105 °C until the spots are clearly developed, and examine under daylight.

[0044] 2.2 Preparation of solutions

[0045] 2.2.1 Preparation of test solution (formula granule test solution)

[0046] Method 1: Take 0.5 g of this product, grind it finely, add 20 ml of 70% methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of 70% methanol to obtain the test solution.

[0047] Method 2: Take 0.5 g of this product, grind it finely, add 20 ml of petroleum ether (30 - 60 °C), ultrasonically treat for 30 minutes, let it stand, discard the supernatant, evaporate the solvent from the residue, add 20 ml of 70% methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of 70% methanol to obtain the test solution.

[0048] Method 3: Take 0.5 g of this product, grind it finely, add 20 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution.

[0049] Method 4: Take 0.5 g of this product, grind it finely, add 20 ml of petroleum ether (30 - 60 °C), ultrasonically treat for 30 minutes, let it stand, discard the supernatant, evaporate the solvent from the residue, add 20 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution.

[0050] Method 5: Take 0.5 g of this product, grind it finely, add 20 ml of ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of ethanol to obtain the test solution.

[0051] Method 6: Take 0.5 g of this product, grind it finely, add 20 ml of petroleum ether (30 - 60 °C), ultrasonically treat for 30 minutes, let it stand, discard the supernatant, evaporate the solvent from the residue, add 20 ml of ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of ethanol to obtain the test solution.

[0052] Method 7: Take 0.5 g of this product, grind it finely, add 20 ml of water to dissolve it, extract with ethyl acetate by shaking 2 times, 20 ml each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution.

[0053] Method 8: Take 0.5 g of this product, grind it finely, add 20 ml of petroleum ether (30 - 60 °C), ultrasonically treat for 30 minutes, let it stand, discard the supernatant, evaporate the solvent from the residue, add 20 ml of water to dissolve it, extract with ethyl acetate by shaking 2 times, 20 ml each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution.

[0054] 2.2.2 Preparation of the control crude drug solution

[0055] Method 1: Take 1 g of the control crude drug of semen aesculi (aesculus wilsonii), add 50 ml of water, heat and decoct for 30 minutes, cool, centrifuge, take the supernatant and concentrate it to about 20 ml, extract with ethyl acetate by shaking 2 times, 20 ml each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the control crude drug solution.

[0056] Method 2: Take 1 g of the control crude drug of semen aesculi (aesculus wilsonii), add 40 ml of petroleum ether (30 - 60 °C), ultrasonically treat for 30 minutes, let it stand, discard the supernatant, evaporate the solvent from the residue, add 50 ml of water, heat and decoct for 30 minutes, cool, centrifuge, take the supernatant and concentrate it to about 20 ml, extract with ethyl acetate by shaking 2 times, 20 ml each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the control crude drug solution.

[0057] 2.2.3 Preparation of the negative control solution

[0058] Take 0.5 g of the negative sample without semen aesculi (aesculus wilsonii), and prepare the negative control solution in the same way as the "Method 8" for the test solution.

[0059] 2.3 Investigation on the preparation methods of the test solution and the control crude drug solution

[0060] Take the sample (formula granules, batch number: 23040049) and the control medicinal material of Aesculus chinensis Bunge var. wilsonii (Tien Shih Li) and prepare the test solution and the control medicinal material solution according to the preparation methods of the above eight test solutions and the two control medicinal material solutions. Spot 5 μl of each of the test solution and the control medicinal material solution on the same silica gel G thin layer plate respectively, develop according to the above thin layer chromatography conditions, take out, dry, spray with 10% phosphomolybdic acid ethanol solution, and heat at 105 °C until the spots are clearly developed.

[0061] It can be seen from Figure 1 that for the test solution prepared by Method VIII, the spots are clear and the separation effect is better. Therefore, Method VIII is selected as the sample treatment method for this thin layer identification; for the control medicinal material of Aesculus chinensis Bunge var. wilsonii (Tien Shih Li) (Method II), the spots are clearer and more corresponding to the spots of the granules. Therefore, Method II is selected as the control medicinal material treatment method.

[0062] 2.4 Investigation of different sample spotting volumes

[0063] Take the sample (formula granules, batch number: 23040049) and the control medicinal material of Aesculus chinensis Bunge var. wilsonii (Tien Shih Li) and prepare the test solution and the control medicinal material solution according to the preparation methods of the above test solution and the control medicinal material solution. Spot on the same silica gel G thin layer plate respectively according to the above thin layer chromatography conditions, develop, take out, dry, spray with 10% phosphomolybdic acid ethanol solution, and heat at 105 °C until the spots are clearly developed.

[0064] It can be seen from Figure 2 that when the sample spotting volumes of the test solution and the control medicinal material solution are 5 μl, the spots at the corresponding positions in the test solution chromatogram and the control medicinal material chromatogram are clearly corresponding, without other interferences. Therefore, the sample spotting volumes of the test solution and the control medicinal material solution are selected as 5 μl.

[0065] 2.5 Specificity experiment of the sample

[0066] Take the test solution (formula granules, batch number: 23040049), the control medicinal material solution and the negative control solution, spot on the same silica gel G thin layer plate respectively according to the above thin layer chromatography conditions, develop, take out, dry, spray with 10% phosphomolybdic acid ethanol solution, and heat at 105 °C until the spots are clearly developed.

[0067] It can be seen from Figure 3 that the test solution chromatogram of the formula granules of Aesculus chinensis Bunge var. wilsonii (Tien Shih Li) shows spots of the same color at the corresponding positions as the control medicinal material chromatogram, and there is no interference from the negative control. It shows that the thin layer identification method has good specificity.

[0068] 2.6 Investigation of different temperatures

[0069] Apply the test solution (formula granules, batch number: 23040049) and the control crude drug solution on the same silica gel G thin-layer plate. According to the above thin-layer chromatography conditions, develop separately under high temperature (40.0 °C) and low temperature (4.0 °C) conditions. Take out, dry in air, spray with 10% ethanolic phosphomolybdic acid solution, and heat at 105 °C until the spots are clearly developed.

[0070] It can be seen from Figure 4 that under different temperature conditions, the test solution chromatogram of semen euonymi (taishili) formula granules and the chromatogram of the control crude drug show spots of the same color at the corresponding positions, and the separation effects are all good. The experimental results show that temperature has no influence on the thin-layer identification of semen euonymi (taishili) formula granules, indicating that this thin-layer identification method has good durability for different temperatures.

[0071] 2.7 Investigation of different humidities

[0072] Apply the test solution (formula granules, batch number: 23040049) and the control crude drug solution on the same silica gel G thin-layer plate. According to the above thin-layer chromatography conditions, develop separately under different humidity conditions. Take out, dry in air, spray with 10% ethanolic phosphomolybdic acid solution, and heat at 105 °C until the spots are clearly developed.

[0073] It can be seen from Figure 5 that under different humidity conditions, the test solution chromatogram of semen euonymi (taishili) formula granules and the chromatogram of the control crude drug show spots of the same color at the corresponding positions, and the separation effects are all good. The experimental results show that humidity has no influence on the thin-layer identification of semen euonymi (taishili) formula granules, indicating that this thin-layer identification method has good durability for different humidities.

[0074] 2.8 Investigation of thin-layer plates from different manufacturers

[0075] Apply the test solution (formula granules, batch number: 23040049) and the control crude drug solution on silica gel G thin-layer plates from different manufacturers. According to the above thin-layer chromatography conditions, develop separately. Take out, dry in air, spray with 10% ethanolic phosphomolybdic acid solution, and heat at 105 °C until the spots are clearly developed.

[0076] It can be seen from Figure 6 that under the conditions of thin-layer plates from different manufacturers, the test solution chromatogram of semen euonymi (taishili) formula granules and the chromatogram of the control crude drug show spots of the same color at the corresponding positions, and the separation effects are all good. The experimental results show that the manufacturer of the thin-layer plate has no significant influence on the thin-layer identification of semen euonymi (taishili) formula granules, indicating that this thin-layer identification method has good durability for thin-layer plates from different manufacturers.

[0077] 2.9 Determination of the thin-layer identification method

[0078] According to the above research results, the thin-layer identification method for semen euonymi (taishili) formula granules is determined as follows:

[0079] Take 0.5 g of this product, grind it finely, add 20 ml of petroleum ether (30 - 60 °C), ultrasonically treat for 30 minutes, let it stand, discard the supernatant, dry the residue by evaporating the solvent, add 20 ml of water to dissolve it, extract with ethyl acetate by shaking 2 times, 20 ml each time, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution. Separately take 1 g of the control crude drug of semen aesculi (aesculus wilsonii), add 40 ml of petroleum ether (30 - 60 °C), ultrasonically treat for 30 minutes, let it stand, discard the supernatant, dry the residue by evaporating the solvent, add 50 ml of water, heat and decoct for 30 minutes, cool, centrifuge, take the supernatant and concentrate it to about 20 ml, extract with ethyl acetate by shaking 2 times, 20 ml each time, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the control crude drug solution. According to the test method of thin-layer chromatography (General Principles 0502, Volume IV, Chinese Pharmacopoeia 2020 Edition), respectively draw 5 μl of the above-mentioned test solution and control crude drug solution, spot them on the same silica gel G thin-layer plate, use toluene - ethyl acetate - methanol - formic acid (10:2:2:0.5) as the developing agent, develop, take out, dry in air, spray with 10% ethanolic solution of phosphomolybdic acid, heat at 105 °C until the spots are clearly visible. In the chromatogram of the test sample, at the corresponding positions to the chromatogram of the control crude drug, spots of the same color appear.

[0080] 2.10 TLC Identification of Different Batches of Semen Aesculi (Aesculus Wilsonii) Formula Granules

[0081] Take the test solutions and control crude drug solutions prepared from different batches of semen aesculi (aesculus wilsonii) formula granules according to the preparation method of the above-mentioned test solution, spot them on the same silica gel G thin-layer plate respectively, develop, take out, dry in air, spray with 10% ethanolic solution of phosphomolybdic acid, heat at 105 °C until the spots are clearly visible.

[0082] It can be seen that Figure 7 in the chromatogram of semen aesculi (aesculus wilsonii) formula granules and the chromatogram of the control crude drug, spots of the same color appear at the corresponding positions.

[0083] 3. TLC Comparison of Semen Aesculi (Aesculus Hippocastanum), Semen Aesculi (Aesculus Chinensis var. Chekiangensis) and Semen Aesculi (Aesculus Wilsonii)

[0084] Respectively take 0.5 g of semen aesculi (Aesculus chinensis Bunge) formula granules, semen aesculi (Aesculus chinensis var. chekiangensis) formula granules, and semen aesculi (Aesculus wilsonii) formula granules, grind them finely, add 20 ml of petroleum ether (30 - 60 °C), ultrasonically treat for 30 minutes, let stand, discard the supernatant, evaporate the solvent from the residue, add 20 ml of water to dissolve it, extract with ethyl acetate by shaking 2 times, 20 ml each time, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution. Additionally, take 1 g of the semen aesculi (Aesculus wilsonii) reference medicinal material, add 40 ml of petroleum ether (30 - 60 °C), ultrasonically treat for 30 minutes, let stand, discard the supernatant, evaporate the solvent from the residue, add 50 ml of water, heat and decoct for 30 minutes, cool, centrifuge, take the supernatant and concentrate it to about 20 ml, extract with ethyl acetate by shaking 2 times, 20 ml each time, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the reference medicinal material solution. According to the thin-layer chromatography method (General Principles 0502, Volume IV of the Chinese Pharmacopoeia 2020 Edition) for testing, respectively pipette 5 μl of the above-mentioned test solution and reference medicinal material solution, and spot them on the same silica gel G thin-layer plate. Use toluene - ethyl acetate - methanol - formic acid (10:2:2:0.5) as the developing solvent, develop, take out, air dry, spray with 10% ethanolic solution of phosphomolybdic acid, and heat at 105 °C until the spots are clearly visible.

[0085] It can be seen from Figure 8 the results that there are spots at the Rf value of 0.3 (the position of the blue frame) for the semen aesculi (Aesculus chinensis Bunge) formula granules and the semen aesculi (Aesculus chinensis var. chekiangensis) formula granules, while there are no spots at the Rf value of 0.3 (the position of the blue frame) for the semen aesculi (Aesculus wilsonii) formula granules. Therefore, this thin-layer method can identify the semen aesculi (Aesculus chinensis Bunge) formula granules, the semen aesculi (Aesculus chinensis var. chekiangensis) formula granules, and the semen aesculi (Aesculus wilsonii) formula granules.

[0086] 4. Thin-layer chromatography comparison of semen aesculi (Aesculus wilsonii) with Aesculus hippocastanum L. and Aesculus wangii Hu

[0087] Take 0.5 g each of the formula granules of Aesculi Semen (Aesculus wilsonii), the formula granules of Aesculus hippocastanum, and the formula granules of Aesculus wangii, grind them finely, add 20 ml of petroleum ether (30 - 60 °C), ultrasonically treat for 30 minutes, let stand, discard the supernatant, evaporate the solvent from the residue, add 20 ml of water to dissolve it, extract twice with ethyl acetate by shaking, 20 ml each time, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution. Additionally, take 1 g of the reference medicinal material of Aesculi Semen (Aesculus wilsonii), add 40 ml of petroleum ether (30 - 60 °C), ultrasonically treat for 30 minutes, let stand, discard the supernatant, evaporate the solvent from the residue, add 50 ml of water, heat and decoct for 30 minutes, cool, centrifuge, take the supernatant and concentrate it to about 20 ml, extract twice with ethyl acetate by shaking, 20 ml each time, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the reference medicinal material solution. According to the test of thin-layer chromatography (General Principles 0502, Volume IV of the Chinese Pharmacopoeia 2020 Edition), absorb 5 μl each of the above-mentioned test solution and reference medicinal material solution, respectively spot them on the same silica gel G thin-layer plate, use toluene - ethyl acetate - methanol - formic acid (10:2:2:0.5) as the developing agent, develop, take out, air dry, spray with 10% ethanolic solution of phosphomolybdic acid, and heat at 105 °C until the spots are clearly visible.

[0088] It can be seen from Figure 9 the results that there is a spot at an Rf value of 0.45 (position of the purple frame) for the formula granules of Aesculus hippocastanum and no spot at an Rf value of 0.7 (position of the red frame), while there is no spot at an Rf value of 0.45 (position of the purple frame) for the formula granules of Aesculi Semen (Aesculus wilsonii) and there is a spot at an Rf value of 0.7 (position of the red frame). Therefore, this thin-layer method can identify the formula granules of Aesculi Semen (Aesculus wilsonii) and the formula granules of Aesculus hippocastanum; there is a spot at an Rf value of 0.3 (position of the blue frame) for the formula granules of Aesculus wangii, while there is no spot at an Rf value of 0.3 (position of the blue frame) for the formula granules of Aesculi Semen (Aesculus wilsonii). Therefore, this thin-layer method can identify the formula granules of Aesculi Semen (Aesculus wilsonii) and the formula granules of Aesculus wangii.

[0089] Comparative Example 1

[0090] "Thin-Layer Chromatographic Identification of Aesculi Semen" Authors: Du Xianghong, Lei Liucheng, Li Ping, Chen Yumei, Jiang Lifang

[0091] 1. Developing agent: n-butanol (saturated with water) - ethyl acetate - acetic acid - water (4:1:1:0.5), for the specific method, see the comparative literature. The results of detecting the Aesculi Semen granules are as Figure 10 shown.

[0092] 2. Developing agent: chloroform - methanol (4:1), for the specific method, see the comparative literature. The results of detecting the Aesculi Semen granules are as Figure 11 shown.

[0093] Conclusion: When using the thin-layer identification method for Chinese medicine semen aesculi in the literature to identify the formula granules, the spots are few and unclear. Therefore, the method in the literature is not applicable to the thin-layer identification of semen aesculi formula granules.

[0094] 3. Identification of aesculin. For the specific method, please refer to the comparative literature. Detect semen aesculi granules and aesculin reference substance.

[0095] Conclusion: It can be seen from Figure 12 that aesculin cannot be detected in semen aesculi formula granules. Therefore, the identification method for aesculin in the literature is not applicable to semen aesculi formula granules.

[0096] 4. Developing agent: ethyl acetate - methanol - formic acid (10:1:0.5). Other conditions are the same as those in the section of determination of thin-layer identification method in 2.9 above. The results are shown in Figure 13 .

[0097] Conclusion: It can be seen from Figure 13 that when using this developing agent, the spots are few and unclear. Therefore, this method is not applicable to the thin-layer identification of semen aesculi formula granules.

Claims

1. A thin-layer identification method for the medicinal material of Aesculus chinensis Bunge or its formula granules, characterized in that: It includes the following steps: The semen aesculi medicinal material or its formula granules and the semen aesculi control medicinal material are first extracted with petroleum ether. After discarding the petroleum ether, they are dissolved or decocted with water and then extracted with ethyl acetate to obtain the test solution and the control medicinal material solution; The control medicinal material solution and the test solution are respectively spotted on a thin-layer plate. After developing with the developing agent, a color-developing agent is sprayed, heated until the spots are clearly developed, and compared and identified under daylight.

2. The thin layer identification method of the medicinal material of Sphaerocarpus ovata or its formulated granules according to claim 1, characterized in that: Specifically, it includes the following steps: (1) Take the semen aesculi control medicinal material, add petroleum ether, shake or ultrasonicate, let it stand, discard the supernatant, dry the residue by evaporating the solvent, add water, heat and decoct, cool, centrifuge, take the supernatant and concentrate it, extract with ethyl acetate by shaking or ultrasonication, evaporate the ethyl acetate extract to dryness, and dissolve the residue in an organic solvent to obtain the control medicinal material solution; (2) Take the semen aesculi medicinal material, add petroleum ether, shake or ultrasonicate, let it stand, discard the supernatant, dry the residue by evaporating the solvent, add water, heat and decoct, cool, centrifuge, take the supernatant and concentrate it, extract with ethyl acetate by shaking or ultrasonication, evaporate the ethyl acetate extract to dryness, and dissolve the residue in an organic solvent to obtain the medicinal material test solution; Or, take the semen aesculi formula granules, add petroleum ether, shake or ultrasonicate, let it stand, discard the supernatant, dry the residue by evaporating the solvent, dissolve it in water, extract with ethyl acetate by shaking or ultrasonication, evaporate the ethyl acetate extract to dryness, and dissolve the residue in an organic solvent to obtain the formula granule test solution; (3) Take the control medicinal material solution and the test solution and respectively spot them on a thin-layer plate. After developing with the developing agent, spray a color-developing agent, heat until the spots are clearly developed, and compare and identify under daylight.

3. The TLC identification method of the semen aesculi medicinal material or its formula granules according to claim 2, characterized in that: The order of steps (1) and (2) can be interchanged.

4. The TLC identification method of the semen aesculi medicinal material or its formula granules according to claim 2, characterized in that: The organic solvent includes alcohol solvents.

5. The thin layer identification method of the medicinal material of Sphaerocarpa sutchuenensis or its formulated granules according to any one of claims 1 or 2, characterized in that: The mass-to-volume ratio of the semen aesculi medicinal material, the semen aesculi control medicinal material or the semen aesculi formula granules to petroleum ether is 1:10 g / mL to 100 g / mL.

6. The thin-layer identification method of the semen aesculi medicinal material or its formula granules according to any one of claims 1 or 2, characterized in that: The developing agent is a mixed solution of toluene, ethyl acetate, methanol and formic acid.

7. The thin-layer identification method of the semen aesculi medicinal material or its formula granules according to any one of claims 1 or 2, characterized in that: The volume ratio of toluene - ethyl acetate - methanol - formic acid in the developing agent is 8 - 12:1 - 3:1 - 3:0.2 - 0.

8.

8. The thin-layer identification method of the semen aesculi medicinal material or its formula granules according to any one of claims 1 or 2, characterized in that: The color-developing agent is a 5 - 15% ethanol solution of phosphomolybdic acid.

9. The thin layer identification method of the medicinal material of Sphaerocarpa sutchuenensis or its formulated granules according to any one of claims 1 or 2, characterized in that: The thin-layer plate is a silica gel G thin-layer plate.

10. The TLC identification method of the semen aesculi medicinal material or its formula granule according to any one of claims 1 or 2, characterized in that: The heating until the spots are clearly developed is heating at 80 - 150 °C until the spots are clearly developed.

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